Journal: Gels

Article Title: A 3D Collagen–Alginate Hydrogel Model for Mechanoregulation of Autophagy in Periodontal Ligament Cells

doi: 10.3390/gels12010091

Figure Lengend Snippet: 3D compression promotes autophagy of PDLCs. ( A ) qRT-PCR analysis for quantification of ATG7 expression at the indicated time points. ( B ) qRT-PCR analysis for quantification of Beclin1 expression at the indicated time points. ( C ) Representative immunofluorescence images of LC3 staining at different time points. Scale bar: 100 μm. ( D ) Ratio of LC3-II/LC3-I quantified at different time points. ( E ) Immunoblotting analysis of P62 protein levels over time. ( F ) Densitometric quantification of P62 normalized to GAPDH. For ( A , B , D , F ), **, ***, ****, and ns indicate p < 0.01, p < 0.001, p < 0.0001 and no significant difference by one-way ANOVA, respectively. Shown as the mean ± SD.

Article Snippet: Samples were incubated with LC3 primary antibody (1:200) overnight at 4 °C, followed by secondary antibody and DAPI staining (Boster).

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Western Blot

Journal: Gels

Article Title: A 3D Collagen–Alginate Hydrogel Model for Mechanoregulation of Autophagy in Periodontal Ligament Cells

doi: 10.3390/gels12010091

Figure Lengend Snippet: AKT-mTOR signaling links compression to autophagy in PDLCs. Compression is transmitted through AKT-mTOR signaling. Modulation of this pathway by the AKT activator SC79 and the mTOR pathway modulator 3BDO alters autophagy-related gene expression. Changes in Beclin1, P62, ATG7, ATG5 and LC3-II reflect the formation of autolysosomes and the overall level of autophagic activity in periodontal ligament cells under compression. The light red arrows indicate the increase in the levels of AKT and mTOR.

Article Snippet: Samples were incubated with LC3 primary antibody (1:200) overnight at 4 °C, followed by secondary antibody and DAPI staining (Boster).

Techniques: Gene Expression, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement C1s cleaves PfEMP1 at interdomain conserved sites inhibiting Plasmodium falciparum cytoadherence.

doi: 10.1073/pnas.2104166118

Figure Lengend Snippet: Fig. 1. Serum fractions B11 and C3 inhibit IT4var19 IE binding to brain endothelial cells. (A) Chromatographic fractionation of serum by size exclusion chromatography. IgG-depleted serum was run on a Superdex 200 Increase 10/300 GL column. Protein fractions were eluted using a linear gradient and the absorbance (mAbs) monitored at 280 nm. The arrows show the binding inhibitory fractions B11 and C3. (B) Binding of IT4var19 IEs to HBEC-5i in binding medium with 10% human serum, IgG-depleted human serum, or serum fractions (A6-C6). A no-serum control was included. Each data point is from two independent experiments done in duplicate, and the mean and SEM are shown for each fraction. Fractions B11 and C3 inhibited the binding the most. These fractions were run on MS/MS, and the components were run to determine their effect on binding as shown in C to identify potential inhibitors for binding. (C) Binding of IT4var19 IEs to HBEC-5i in binding medium with proteins identified by MS/MS in the serum inhibitory B11 and C3 fractions (attractin, C1s, C4b, factor H, IgD, ITIH1, ITIH2, and kappa light chain). Data are mean ± SEM, and each data point is from an independent experiment. **P < 0.01; ***P < 0.001.

Article Snippet: For binding inhibition assays, serum and serum fractions (at 10%), recombinant human attractin (7238-AT-050; Novus Biologicals), C1s (A104; Complement Technology; or 2060-SE; R&D Systems), human complement C4b (H00000721-Q01; Novus Biologicals), human complement factor H (H00003075-P03; Novus Biologicals), IgD (NB100-62667; Novus Biologicals), human kappa light chain (H00003514-P01; Novus Biologicals), human inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1) (H00003697-P01; Novus Biologicals), and H2 (ITIH2) (NBP2-31750PEP; Novus Biologicals) (at physiological concentrations in serum, ∼10% of the physiological concentrations or an estimated concentration where serum levels could not be found; see SI Appendix, Table S1) were added to the parasites in DMEM binding medium immediately before coincubation with HBEC-5i.

Techniques: Binding Assay, Fractionation, Size-exclusion Chromatography, Control, Tandem Mass Spectroscopy

Journal: Oncology Letters

Article Title: Role of CYP4F2 as a novel biomarker regulating malignant phenotypes of liver cancer cells via the Nrf2 signaling axis

doi: 10.3892/ol.2020.11874

Figure Lengend Snippet: CYP4F2 overexpression inhibits the Nrf2 signaling pathway in liver cancer cells. (A) Reverse transcription-quantitative PCR was used to determine the Nrf2 mRNA expression levels in NC, Hep3B+vector and Hep3B+CYP4F2. (B) Western blot analysis was used to determine the protein expression levels in 5 genes and the results were quantified for CYP4F2 (C), Nrf2 (D), NQO1 (E), HO-1 (F) and FTH1 (G). Data are presented as the mean ± SD. One-way ANOVA was used with the Bonferroni correction. *P<0.05; **P<0.01; ***P<0.001. There is no significant difference between NC and Hep3B+vector in all figures. Hep3B cells were transfected with lentivirus to overexpress CYP4F2 (Hep3B+CYP4F2), or transfected with control lentivirus was used as a negative control (Hep3B+vector), and untreated Hep3B cells served as normal control (NC).

Article Snippet: Subsequently, the membrane was blocked using 5% skimmed milk and incubated overnight at 4°C with primary antibodies against CYP4F2 (cat. no. AF9051; 1:1,000; Affinity Biosciences), Nrf2 (cat. no. 66504-1-Ig; 1:1,000; ProteinTech Group, Inc.), NQO1(cat. no. 67240-1-Ig; 1:5,000; ProteinTech Group, Inc.), HO-1 (cat. no. 66743-1-Ig; 1:1,000; ProteinTech Group, Inc.) and FTH1 (cat. no. 10727-1-AP; 1:1000), GAPDH (cat. no. BA2913; 1:500; Wuhan Boster Biological Technology, Ltd.) as the internal reference.

Techniques: Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Western Blot, Transfection, Control, Negative Control